This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic-projects "CENAKVA" No. CZ.1.05/2.1.00/01.0024 and "CENAKVA II'' No. LO1205 under the National Sustainability Program I, by the Grant Agency of the University of South Bohemia in &#268;esk&#233; Bud&#283;jovice (012/2016/Z), and by the Grant Agency of the Czech Republic (No. 16-06498S)

1. Crayfish Selection
<ol>
	<li>In order to successfully apply the current approach to crayfish, select the respective adult specimens with sufficient carapace sizes (which is a carapace length of at least 30 mm) for sensor attachment, visually examine it for the absence of diseases, and check whether it lifts both chelae when it is touched. The above-mentioned parameters indicate an eligible state of crayfish health. 
	NOTE: If several crayfish are expected to be used in the trial and are exposed to the same conditions, the experimental group should be formed based on several parameters: (i) similar weight and length; (ii) comparable heart rate; (iii) pronounced nocturnal activity; (iv) regular food consumption; (v) inter-molting period17. Sometimes, it is hard to define whether a crayfish is near to molting by the heart rate measurements or visual or tactile examinations only; therefore, the analyses of the crayfish&#39;s hemolymph total protein content can be helpful. Protein content is expected to be higher when the crayfish is closer to molting than in the inter-molting state18.</li>
</ol>
2. Recording of Crayfish Cardiac Activity and Behavior
<ol>
	<li>In order to noninvasively measure crayfish heart rates, preliminarily prepare the sensor for this procedure. Before this, put a crayfish into the tank with water and let it acclimate there for a few days as the preparation of the sensor19 will also take a few days.
	<ol>
		<li>Axially couple an IR light-emitting diode (LED) with a phototransistor. Attach the optical sensor circuit onto a board; it will require a power supply of 5 V. For the LED connection, place a 200 &#937; resistor on the IR sensor board; in order to connect the phototransistor, place a 220 &#937; resistor on the board.</li>
		<li>When attached to the crayfish, the sensor output is modulated by the amount of hemolymph filling the crayfish cardiac muscle and scatters an incident light from the LED. In order to avoid reciprocal interference of the illuminated IR light by the LED and the reflected IR light from the crayfish heart, which is received by the phototransistor, place a small wall (0.5 x 1.5 x 4 mm, thickness x height x width) made of black antistatic plastic between the LED and the phototransistor.</li>
		<li>Place the LED in a waterproof package, and cover the surface of the sensor with the waterproof dielectric gel from the side adjacent to the carapace for the protection of the electronic components from potential damage (Figure 1). Let the gel dry for 3 days in order to gain its best protective properties.</li>
		<li>For an analog signal, attach thin flexible cables (about 3 m long) to the sensor and connect to the analogue-to-digital converter (ADC); from this, a digitized signal will be transferred to a personal computer over a USB interface, at which point the information about the crayfish cardiac activity is saved, analyzed in real-time with special software (see Table of Materials), and stored for further detailed analyses.</li>
	</ol>
	</li>
	<li>As soon as the sensor is prepared, attach it to the crayfish. In order to do this, switch the computer on and run the software. Determine the number of crayfish to be fixed to the sensors and recorded heart rate to be saved to the date file.</li>
	<li>Remove the crayfish from the water and wipe its dorsal carapace side with a paper towel. Wrap the chelae and abdomen of the crayfish in the paper towel in order to avoid any damages by human hand and to eliminate additional stress on the crayfish caused by warm human hands. 
	NOTE: Do not use a previous cooling of the crayfish on ice or in the freezer for its immobilization before manipulations with the sensor attachment. The difference in temperatures leads to crayfish dorsal surface weeping which, in turn, leads to unreliable sensor fastening and quick adhesive detachment from the crayfish&#39;s carapace.</li>
	<li>Prepare a surface (i.e., take a small flat piece of plastic or tear a piece of sticky tape and fix it to a table) and a stick for mixing the glue. Press out two small drops (of a diameter of about 0.5 cm) from tubes A and B containing epoxy glue and quickly mix them.</li>
	<li>Attach the sensor to the crayfish dorsal carapace and try to find the place in which the cardiac signal amplitude would be maximal. Hold the crayfish with the sensor in one hand and, using the other free hand, put a drop of mixed glue on each of the four auxiliary wires located on the sensor (fix them in between steps 2.1.1 and 2.1.4.). Do not move the sensor at least 5 min until the glue hardens (the glue hardening depends on the ambient temperature and humidity). 
	NOTE: When fixing the sensor to the crayfish carapace, examine thoroughly the whole cardiac area from the carapace side in order to define the area with the best (maximal) cardiac signal amplitude. That will help the software to provide more precise heart rate calculations.</li>
	<li>Touch the glue using a free hand, and if it is not sticky, put the unwrapped crayfish with the attached sensor (Figure 2) to the box without water for few more minutes until the glue is completely dry. 
	NOTE: An optimal temperature for crayfish and glue manipulation varies from 18 to 22 &#176;C. At these temperatures, the glue hardens within 5 to 7 min and is completely dry within 8 to 10 min. At lower temperatures, the stress in the crayfish is less pronounced; however, the glue needs more time to harden, about 15 and 20 min under 15 &#176;C and 10 &#176;C, respectively. At higher temperatures, particularly above 25 &#176;C, the glue hardens within 3 min, but the crayfish undergoes much more stress; therefore, try to minimize the exposure of the crustacean to extreme conditions without water.</li>
	<li>Before moving the crayfish back into the tank, dip its cephalothorax into the water several times with short intervals of a few seconds in order to allow a discharge of the air that has accumulated in the gills, and leave the crayfish in the water for approximately 1 h to remove any excess chemicals. After this process is complete, release the crayfish into the water and allow it to acclimate for one to two weeks under experimental conditions, depending on observed physiological indices. Optimal water exchange during the acclimation periods is every other day. 
	NOTE: Characteristics of crayfish that have acclimated and are in a healthy state include pronounced circadian cardiac and locomotor activities, regular food consumption, and spending most daylight in a specialized shelter (if provided).</li>
</ol>
3. Camera and Software Setup
<ol>
	<li>Start the software; the video camera will automatically switch on.</li>
	<li>Select an option of movement detection, thoroughly detect area of each tank on the screen&#160;and the software will start tracking the behavior and linking it with the cardiac activity recordings. 
	NOTE: A crayfish motion detection module consists of a video camera that tracks crayfish behavior from below the tank and the software that combines the behavior with cardiac activity. The data from the module are used to facilitate more precise cardiac activity data processing by eliminating periods in which the crayfish demonstrates high locomotive activity. Sudden crayfish movements (i.e., an escape reaction or feeding initiation) can result in fluctuations or short-time spikes in cardiac signals that may reduce the precision of cardiac interval calculations.</li>
</ol>

<ol>
	<li>Bownik, A., Soko&#322;owska, N., &#346;laska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+apomorphine,+a+dopamine+agonist,+on+Daphnia+magna:+Imaging+of+swimming+track+density+as+a+novel+tool+in+the+assessment+of+swimming+activity.">Effects of apomorphine, a dopamine agonist, on Daphnia magna: Imaging of swimming track density as a novel tool in the assessment of swimming activity.</a> Science of the Total Environment. 635, 249-258 (2018).</li><li>Jeong, T. Y., Yoon, D., Kim, S., Kim, H. Y., Kim, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+characterization+for+adverse+effect+of+propranolol+in+Daphnia+magna.+based+on+behavior+and+physiology+monitoring+and+metabolite+profiling.">Mode of action characterization for adverse effect of propranolol in Daphnia magna. based on behavior and physiology monitoring and metabolite profiling.</a> Environmental Pollution. 233, 99-108 (2018).</li><li>do Nascimento, M. T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+water+quality,+toxicity+and+estrogenic+activity+in+a+nearshore+marine+environment+in+Rio+de+Janeiro,+Southeastern+Brazil.">Determination of water quality, toxicity and estrogenic activity in a nearshore marine environment in Rio de Janeiro, Southeastern Brazil.</a> Ecotoxicology and Environmental Safety. 149, 197-202 (2018).</li><li>Xiao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+quality+monitoring+using+abnormal+tail-beat+frequency+of+crucian+carp.">Water quality monitoring using abnormal tail-beat frequency of crucian carp.</a> Ecotoxicology and Environmental Safety. 111, 185-191 (2015).</li><li>Aagaard, A., Andersen, B. B., Depledge, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+monitoring+of+physiological+and+behavioral+activity+in+marine+organisms+using+non-invasive,+computer+aided+techniques.">Simultaneous monitoring of physiological and behavioral activity in marine organisms using non-invasive, computer aided techniques.</a> Marine Ecology Progress Series. 73 (2), 277-282 (1991).</li><li>Bloxham, M. J., Worsfold, P. J., Depledge, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+biological+and+chemical+monitoring:+in+situ.+physiological+responses+of+freshwater+crayfish+to+fluctuations+in+environmental+ammonia+concentrations.">Integrated biological and chemical monitoring: in situ. physiological responses of freshwater crayfish to fluctuations in environmental ammonia concentrations.</a> Ecotoxicology. 8 (3), 225-237 (1999).</li><li>Depledge, M. H., Andersen, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-aided+physiological+monitoring+system+for+continuous,+long-term+recording+of+cardiac+activity+in+selected+invertebrates.">A computer-aided physiological monitoring system for continuous, long-term recording of cardiac activity in selected invertebrates.</a> Comparative Biochemistry and Physiology. A, Comparative Physiology. 96 (4), 473-477 (1990).</li><li>Depledge, M. H., Galloway, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthy+animals,+healthy+ecosystems.">Healthy animals, healthy ecosystems.</a> Frontiers in Ecology and the Environment. 3 (5), 251-258 (2005).</li><li>Holdich, D. M., Reynolds, J. D., Souty-Grosset, C., Sibley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+ever+increasing+threat+to+European+crayfish+from+non-indigenous+crayfish+species.">A review of the ever increasing threat to European crayfish from non-indigenous crayfish species.</a> Knowledge and Management of Aquatic Ecosystems. 11, 394-395 (2009).</li><li>Vogt, G., Holdich, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy.">Functional anatomy.</a> Biology of freshwater crayfish. , 53-151 (2002).</li><li>Bierbower, S. M., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+heart+and+ventilatory+rates+in+freely+moving+crayfish.">Measures of heart and ventilatory rates in freely moving crayfish.</a> Journal of Visualized Experiments. (32), e1594 (2009).</li><li>Li, H., Listerman, L. R., Doshi, D., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+in+blind+cave+crayfish+during+environmental+disturbances+and+social+interactions.">Heart rate in blind cave crayfish during environmental disturbances and social interactions.</a> Comparative Biochemistry and Physiology Part A: Molecular &amp; Integrative Physiology. 127 (1), 55-70 (2000).</li><li>Listerman, L. R., Deskins, J., Bradacs, H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+within+male+crayfish:+social+interactions+and+effects+of+5-HT.">Heart rate within male crayfish: social interactions and effects of 5-HT.</a> Comparative Biochemistry and Physiology Part A: Molecular &amp; Integrative Physiology. 125 (2), 251-263 (2000).</li><li>Burnett, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+noninvasive+method+for+measuring+heartbeat+of+intertidal+animals.">An improved noninvasive method for measuring heartbeat of intertidal animals.</a> Limnology and Oceanography: Methods. 11 (2), 91-100 (2013).</li><li>Fedotov, V. P., Kholodkevich, S. V., Strochilo, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+contractile+activity+of+the+crayfish+heart+with+the+aid+of+a+new+non-invasive+technique.">Study of contractile activity of the crayfish heart with the aid of a new non-invasive technique.</a> Journal of Evolutionary Biochemistry and Physiology. 36 (3), 288-293 (2000).</li><li>Kholodkevich, S. V., Ivanov, A. V., Kurakin, A. S., Kornienko, E. L., Fedotov, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+biomonitoring+of+surface+water+toxicity+level+at+water+supply+stations.">Real time biomonitoring of surface water toxicity level at water supply stations.</a> Environmental Bioindicators. 3 (1), 23-34 (2008).</li><li>Kuznetsova, T. V., Sladkova, S. V., Kholodkevich, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+functional+state+of+crayfish+Pontastacus+leptodactylus+in+normal+and+toxic+environment+by+characteristics+of+their+cardiac+activity+and+hemolymph+biochemical+parameters.">Evaluation of functional state of crayfish Pontastacus leptodactylus in normal and toxic environment by characteristics of their cardiac activity and hemolymph biochemical parameters.</a> Journal of Evolutionary Biochemistry and Physiology. 46 (3), 241-250 (2010).</li><li>Sladkova, S. V., Kholodkevich, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+protein+in+hemolymph+of+crawfish+Pontastacus+leptodactylus+as+a+parameter+of+the+functional+state+of+animals+and+a+biomarker+of+quality+of+habitat.">Total protein in hemolymph of crawfish Pontastacus leptodactylus as a parameter of the functional state of animals and a biomarker of quality of habitat.</a> Journal of Evolutionary Biochemistry and Physiology. 47 (2), 160-167 (2011).</li><li>Pautsina, A., Kuklina, I., &#352;tys, D., C&#237;sa&#345;, P., Koz&#225;k, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+crayfish+cardiac+activity+monitoring+system.">Noninvasive crayfish cardiac activity monitoring system.</a> Limnology and Oceanography: Methods. 12 (10), 670-679 (2014).</li><li>C&#237;sa&#345;, P., Saberioon, M., Koz&#225;k, P., Pautsina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+contactless+system+for+crayfish+heartbeat+monitoring:+Undisturbed+crayfish+as+bio-indicator.">Fully contactless system for crayfish heartbeat monitoring: Undisturbed crayfish as bio-indicator.</a> Sensors and Actuators B: Chemical. 255, 29-34 (2018).</li></ol>

The authors have nothing to disclose.

It has been widely suggested that the measurement of certain physiological parameters (such as heart or ventilation rate or both) is a more reliable method for recording crayfish reactions than the evaluation of behavioral responses that do not always occur immediately11. However, it is evident that the most efficient approach for assessing real crayfish reactions to environmental changes is the combination of cardiac activity and behavior recordings since that makes it possible to see the reason(...

The subject of aquatic organisms&#39; applications, both as model organisms for various laboratory investigations1,2 and as tools for monitoring industrial and natural/environmental water quality3,4, appears to be well studied. Nevertheless, this topic is still of noteworthy interest for humans, irrespective of whether they belong to the scientific community or to other occupations. In spite of the existence of a number of advanced methods for monitoring certain parameters (so-called &#34;biomarkers&#34;)5,6,7,8, the most important requirements for selecting an indicator consist of three simple factors: (i) simplicity, (ii) reliability, and (iii) general availability.
Crayfish, as an essential representative of freshwater fauna, distinguishes itself because it is found worldwide, is widespread, and, in most cases9, has a sufficiently large and hard carapace suitable for manipulation. This crustacean belongs to the group of higher invertebrates that provide sufficient development of vital physiological systems and respective organs while, at the same time, maintaining a relatively simple organization10.
Methods based on the assessment of the range of crayfishes&#39; biological and/or behavioral parameters, as described in the scientific literature, have significantly contributed to the development of biomonitoring and crayfish studies in general. Most of the currently available invasive methods for crayfish heart rate measurements are based on electrocardiogram recordings that require a complex and precise surgical procedure11,12,13; such manipulations can cause significant stress to and may require prolonged adaptation by the crayfish. Also, it is not known how long a crayfish can carry such electrodes and whether it will successfully molt while carrying such an attachment. The described noninvasive methods are based on plethysmographic recordings, which are complicated by hardware complexity and require a conditioning circuit for signal filtering14 and an amplification or precise and expensive optic components15,16.
In this study, we described an approach that contributes to existing results and offers new alternatives for improving current crayfish heart rate measurement procedures. Among the advantages, there are (i) a fast and noninvasive attachment that does not require a prolonged physiological adaptation; (ii) crayfishes&#39; capability to carry the sensor within a period of a few months from molting to molting; (iii) the software capable of monitoring real-time cardiac and behavioral activities and the evaluation of data obtained concurrently from multiple crayfish; (iv) a low manufacturing price and simplicity. The biomonitoring system that we describe permits the noninvasive and continuous monitoring of crayfish cardiac and locomotor activities based on changes in crayfishes&#39; etho-physiological characteristics. This system can easily be applied in laboratory examinations of the crayfish cardiac physiology and/or ethology, in addition to industrial implementations for controlling water quality at water treatment and supply facilities.

<table><tbody><tr><td>IR LED diode</td><td>KINGBRIGHT ELECTRONIC</td><td>KP-3216F3C</td><td></td></tr><tr><td>Phototransistor</td><td>EVERLIGHT</td><td>ELPT15-21C</td><td></td></tr><tr><td>Resistor</td><td>ROYAL OHM</td><td>0805S8J0201T5E</td><td></td></tr><tr><td>Resistor</td><td>ROYAL OHM</td><td>0805S8F2200T5E</td><td></td></tr><tr><td>Capacitor</td><td>KEMET</td><td>C0805C334K5RACTU</td><td></td></tr><tr><td>Cable</td><td>TECHNOKABEL</td><td>FTP KAT.5E 4X2X0,14C</td><td></td></tr><tr><td>Connector</td><td>HARTING</td><td>21348100380005</td><td></td></tr><tr><td>Connector</td><td>HARTING</td><td>21348000380005</td><td></td></tr><tr><td>Dielectric gel</td><td>KRAYDEN</td><td>Sylgard 535</td><td></td></tr><tr><td>Analogue-to-digital convertor</td><td>TEDIA</td><td>UDAQ-1416CA</td><td></td></tr><tr><td>Glue</td><td>KUPSITO.SK</td><td>7338723044</td><td></td></tr><tr><td>Kinect video camera</td><td>ABCSTORE.CZ</td><td>GT3-00002</td><td></td></tr><tr><td>Analysis software</td><td>University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Institute of Complex Systems</td><td></td><td>Link to the software: www.frov.jcu.cz/crayfishmonitoring&lt;br/&gt; User name: frov&lt;br/&gt; Password: CF2018</td></tr></tbody></table>

continuous noninvasive measuring of crayfish cardiac and behavioral activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

As a result, we obtained a combination of crayfish cardiac and behavioral activities, recorded and saved in a txt-format file (Figure 3). Besides the number of experimental crayfish, the date, and the sampling rate, the file consists of three columns: (1) the continual time in hh:mm:ss format; (2) the heart rate automatically calculated in beats per minute; (3) the locomotion registered as absence (0) or presence (1) of any movement. When the crayfish was ina...

Watch this Scientific Journal Video about Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities at JoVE.com

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

continuous-noninvasive-measuring-crayfish-cardiac-behavioral

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

5.9K Views.  University of South Bohemia in České Budĕjovice. This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

Video: Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Neuroscience

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception 

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Impedance Pneumography for Minimally Invasive Measurement of Heart Rate in Late Stage Invertebrates

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by environmental temperature, this has the potential to alter thermal physiological performance in a variety of marine organisms. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography to understand how cardiac performance in late stage invertebrates changes under acute thermal stress. The protocol presents a minimally invasive technique that allows for real-time collection of heart rate during a temperature ramping experiment. Data are easily manipulated to generate an Arrhenius plot that is used to calculate Arrhenius break temperature (ABT), the temperature at which heart rate begins to decline with increasing temperatures. This technique can be used in a variety of late stage invertebrates (i.e., crabs, mussels, or shrimps). Although the protocol focuses solely on the impact of temperature on cardiac performance, it can be modified to understand the potential for additional stressors (e.g., hypoxia or hypercapnia) to interact with temperature to influence physiological performance. Thus, the method has potential for wide-ranging applications to further understand how marine invertebrates respond to acute changes in the environment.

Measuring heart rate during a thermal challenge provides insight into physiological responses of organisms as a consequence of acute environmental change. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography as a relatively noninvasive and nonlethal approach to measure heart rate in late stage invertebrates.

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by ...

Environment

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Biology

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack.  Although it is not known whether invertebrates posses an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a  sympathetic-like  response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli.  Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state.  Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems.   More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors.

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1.
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students&#8217; practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish&#8217;s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

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Recording Behavioral Responses to Reflection in Crayfish

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

Impedance Pneumography for Minimally Invasive Measurement of Heart Rate in Late Stage Invertebrates

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Recordings of Neural Circuit Activation in Freely Behaving Animals

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern